Journal of Pharmaceutical Analysis

2017, 7(6): 后插1-2.

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Application of HPLC and ESI-MS techniques in the analysis of phenolic acids and flavonoids from green leafy vegetables (GLVs)

Ramesh Kumar Bonta

2017, 7(6): 349-364.

Abstract(148) PDF(9)

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Diets containing high proportions of fruits and vegetables reduce the risk of onset of chronic diseases.The role of herbal medicines in improving human health is gaining popularity over the years,which also increases the need for safety and efficiency of these products.Green leafy vegetables(GLVs)are the richest source of phenolic compounds with excellent antioxidant properties. Increased consumption of diets containing phenolic compounds may give positive and better results to human health and significantly improves the immune system.Highly selective,susceptible and versatile analytical techniques are necessary for extraction,identifica-tion, and quantification of phenolic compounds from plant extracts, which helps to utilize their important biological properties. Recent advances in the pre-treatment procedures, separation techniques and spectro-metry methods are used for qualitative and quantitative analysis of phenolic compounds.The online coupling of liquid chromatography with mass spectrometry(LC–MS)has become a useful tool in the metabolic profiling of plant samples.In this review,the separation and identification of phenolic acids and flavonoids from GLVs by LC–MS have been discussed along with the general extraction procedures and other sources of mass spectrometer used. The review is devoted to the understanding of the structural configuration, nature and accumulation pattern of phenolic acids and flavonoids in plants and to highlighting the recent developments in the chemical investigation of these compounds by chromatographic and spectroscopic techniques.It concludes with the advantages of the combination of these two methods and prospects.

Development of a novel stability indicating RP-HPLC method for quantification of Connexin43 mimetic peptide and determination of its degradation kinetics in biological fluids

Rohit Bisht, Ilva D. Rupenthal, Sreevalsan Sreebhavan, Jagdish K. Jaiswal

2017, 7(6): 365-373.

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Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stability–indicating reversed-phase high performance liquid chromatography(RP-HPLC)method for the quantification of Cx43MP under stress conditions.These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 ℃. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9–250μg/mL(R2≥0.998),and the limits of detection(LOD)and quantification(LOQ) were found to be 0.90 and 2.98 μg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250μg/mL was 96.79%, 98.25% and 99.06% with a RSD of<2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 ℃(0% recovery)and within 12 h at 80 ℃(0.34% recovery).Cx43MP was found to be more stable in bovine vitreous(t1/2slow=171.8 min)compared to human plasma(t1/2slow=39.3 min)at 37 ℃ according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP.

A validated UPLC–MS/MS method for simultaneous determination of imatinib,dasatinib and nilotinib in human plasma

Jing Zeng, Hualin Cai, Zhiping Jiang, Qing Wang, Yan Zhu, Ping Xu, Xielan Zhao

2017, 7(6): 374-380.

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A sensitive, rapid, simple and economical ultra-performance liquid chromatography–tandem mass spectro-metric method (UPLC–MS/MS) was developed and validated for simultaneous determination of imatinib, dasatinib and nilotinib in human plasma using gliquidone as internal standard (IS). Liquid-liquid extraction method with ethyl acetate was used for sample pre-treatment. The separation was performed on an Xtimate Phenyl column using isocratic mobile phase consisting of A (aqueous phase: 0.15% formic acid and 0.05% ammonium acetate)and B(organic phase:acetonitrile)(A:B=40:60,v/v).The flow rate was 0.25 mL/min and the total run time was 6 min. The multiple reaction monitoring (MRM) transitions, m/z 494.5→394.5 for imatinib, 488.7→401.5 for dasatinib, 530.7→289.5 for nilotinib and 528.5→403.4 for IS, were chosen to achieve high selectivity in the simultaneous analyses. The method exhibited great improvement in sensitivity and good linearity over the concentration range of 2.6–5250.0 ng/mL for imatinib, 2.0–490.0 ng/mL for dasatinib,and 2.4–4700.0 ng/mL for nilotinib.The method showed acceptable results on sensitivity,specificity, recovery, precision, accuracy and stability tests. This UPLC–MS/MS assay was successfully used for human plasma samples analysis and no significant differences were found in imatinib steady-state trough concentra-tions among the SLC22A5?1889T>C or SLCO1B3 699G>A genotypes(P>0.05).This validated method can provide support for clinical therapeutic drug monitoring and pharmacokinetic investigations of these three tyrosine kinase inhibitors(TKIs).

Development and validation of a high throughput LC–MS/MS method for simultaneous quantitation of pioglitazone and telmisartan in rat plasma and its application to a pharmacokinetic study

Pinaki Sengupta, Bappaditya Chatterjee, Uttam Kumar Mandal, Bapi Gorain, Tapan Kumar Pal

2017, 7(6): 381-387.

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Management of cardiovascular risk factors in diabetes demands special attention due to their co-existence. Pioglitazone (PIO) and telmisartan (TLM) combination can be beneficial in effective control of cardiovascular complication in diabetes.In this research,we developed and validated a high throughput LC–MS/MS method for simultaneous quantitation of PIO and TLM in rat plasma. This developed method is more sensitive and can quantitate the analytes in relatively shorter period of time compared to the previously reported methods for their individual quantification. Moreover, till date, there is no bioanalytical method available to simultaneously quantitate PIO and TLM in a single run. The method was validated according to the USFDA guidelines for bioanalytical method validation.A linear response of the analytes was observed over the range of 0.005–10μg/mL with satisfactory precision and accuracy. Accuracy at four quality control levels was within 94.27%–106.10%. The intra-and inter-day precision ranged from 2.32% to 10.14% and 5.02% to 8.12%,respectively.The method was reproducible and sensitive enough to quantitate PIO and TLM in rat plasma samples of a preclinical pharmacokinetic study.Due to the potential of PIO-TLM combination to be therapeutically explored,this method is expected to have significant usefulness in future.

Application of a validated HPLC-PDA method for the determination of melatonin content and its release from poly(lactic acid)nanoparticles

Leiziani Gnatkowski Martins, Najeh Maissar Khalil, Rubiana Mara Mainardes

2017, 7(6): 388-393.

Abstract(223) PDF(6)

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Melatonin is a natural hormone and with the advancement of age its production declines and thereby may result in some neurological disorders.Exogenous administration of melatonin has been suggested as a neuroprotective agent. Due to its low oral bioavailability, the loading of melatonin in polymeric nanoparticles could be an important tool to effectively use exogenous melatonin. The quantification of the incorporated drug within polymeric nanoparticles is an important step in nanoparticles characterization.An analytical method using high performance liquid chromatography equipped with photodiode array detector(HPLC-PDA)was developed and validated for melatonin determination in poly(lactic acid)nanoparticles obtained by a single emulsion-solvent evaporation technique.The melatonin in vitro release profile also was determined by the HPLC method.Mobile phase consisted of acetonitrile:water(65:35,v/v)pumped at a flow rate of 0.9 mL/min,in the isocratic mode and PDA detector was set at 220 nm.The method was validated in terms of the selectivity,linearity,precision, accuracy,robustness,limits of detection and quantification.Analytical curve was linear over the concentration range of 10–100 μg/mL, and limits of detection and quantification were 25.9 ng/mL and 78.7 ng/mL, respectively. The mean recovery for melatonin was 100.47% (RSD = 1.25%, n = 9). In the intra- and inter-assay,the coefficient of variation was less than 2%.Robustness was proved performing changes in mobile phase, column temperature and flow rate.The method was suitable for the determination of melatonin encapsulation efficiency in poly(lactic acid)nanoparticles and for the evaluation of melatonin in vitro release profile.

Synthesis,isolation,identification and characterization of new process-related impurity in isoproterenol hydrochloride by HPLC,LC/ESI-MS and NMR

Neeraj Kumar, Subba Rao Devineni, Prasad Reddy Gajjala, Shailendra Kumar Dubey, Pramod Kumar

2017, 7(6): 394-400.

Abstract(127) PDF(8)

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One unknown impurity(Imp-II)during the analysis of laboratory batches of isoproterenol hydrochloride was detected in the level ranging from 0.04% to 0.12% by high performance liquid chromatography with UV detection. The unknown impurity structure was proposed as 4-[2-(propan-2-ylamino)ethyl]benzene-1,2-diol (Imp-Ⅱ)using the liquid chromatography–mass spectrophotometry(LC–MS)analysis.Imp-Ⅱ was isolated by semi-preparative liquid chromatography from the impurity-enriched reaction crude sample. Its proposed structure was confirmed by nuclear magnetic spectroscopy such as 1H, 13C,DEPT(1D NMR),HSQC(2D NMR) and infrared spectroscopy(IR),and retention time and purity with HPLC followed by the chemical synthesis. Due to less removable nature of Imp-II during the purification,the synthetic process was optimized proficiently to control the formation of Imp-II below to the limit<0.12% in the course of reaction.The new chemical route was developed for the preparation of this impurity in required quantity with purity to use as reference standard. The most probable mechanism for the formation of Imp-II was discussed in details.

Quantification of theophylline or paracetamol in milk matrices by high-performance liquid chromatography

Tania A.P. Fernandes, Jo?o P. Aguiar, Ana I. Fernandes, Jo?o F. Pinto

2017, 7(6): 401-405.

Abstract(63) PDF(1)

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A simple, accurate and sensitive high-performance liquid chromatography (HPLC) method was developed, validated and applied to the determination of either theophylline or paracetamol in milk-based samples. The method allowed drug quantification in fresh and powdered milk with a relatively short run time of analysis and it was also successfully applied to the quantification of the drugs in solid dosage forms intended for pediatric use. Moreover, the main significant advantages over other published works are the simplicity of the sample preparation, reduced assay time and sample loss. The method meets the International Conference on Harmonization guideline for analytical methods validation regarding specificity,linearity,accuracy,precision, specificity and robustness as required by health authorities and applied by industry while designing and marketing new drug products.The technique encompasses the separation of the analytes with a reverse phase C18column under isocratic conditions and UV detection at 272 nm and 243 nm,respectively,for theophylline and paracetamol. The lower limit of quantification for both drugs was determined as 0.2μg/mL and the between-batch accuracy was 99.7%.This HPLC method allows quantification of theophylline and paracetamol in milk matrices and it can be applied in the design,development and production of milk-based pediatric dosage forms.

Development of a UPLC–MS/MS method for determination of pimavanserin tartrate in rat plasma:Application to a pharmacokinetic study

Shixiao Wang, Yang Wang, Shuang Gao, Yuanyuan Zhang, Hanpei Wang, Longshan Zhao, Kaishun Bi, Shaojie Wang, Xiaohui Chen

2017, 7(6): 406-410.

Abstract(47) PDF(0)

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A simple, rapid and sensitive method based on an ultra-performance liquid chromatography–tandem mass spectrometry(UPLC–MS/MS)has been developed and validated for the determination of pimavanserin in rat plasma.The analyte was extracted by protein precipitation with methanol and separated on an ACQUITY BEH C18column(100 mm×2.1 mm,1.7μm;Waters,USA),with an isocratic elution of acetonitrile-water containing 10 mM ammonium acetate (70:30, v/v), at a flow rate of 0.2 mL/min for 2.5 min. The analyte and clarithromycin (the internal standard) were detected and quantified in positive ion mode using multiple reaction monitoring transitions at m/z 428.2 → 223.0 for pimavanserin and m/z 748.5 → 589.5 for clarithromycin. Relative coefficient (r) for the calibration curve was more than 0.9980. The intra-day and inter-day precisions(relative standard deviation,RSD%)were less than 13.3% and 10.5%,respectively,and the accuracy(relative error,RE%)was within ± 11.5%.The analytical method was successfully applied to a routine pharmacokinetic study of pimavanserin in rats after oral administration at the dose of 10 mg/kg.

Simultaneous colorimetric determination of morphine and ibuprofen based on the aggregation of gold nanoparticles using partial least square

Morteza Bahram, Tayyebeh Madrakian, Sakineh Alizadeh

2017, 7(6): 411-416.

Abstract(128) PDF(2)

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In this work a new method is presented for simultaneous colorimetric determination of morphine(MOR)and ibuprofen(IBU)based on the aggregation of citrate-capped gold nanoparticles(AuNPs).Citrate-capped AuNPs were aggregated in the presence of MOR and IBU. The difference in kinetics of AuNPs aggregation in the presence of MOR/IBU was used for simultaneous analysis of MOR and IBU. The formation and size of synthesized AuNPs and the aggregated forms were monitored by infra-red(IR)spectroscopy and transmission electron microscopy(TEM),respectively.By adding MOR or IBU the absorbance was decreased at 520 nm and increased at 620 nm.The difference in kinetic profiles of aggregation was applied for simultaneous analysis of MOR and IBU using partial least square(PLS)regression as an efficient multivariate calibration method.The number of PLS latent variables was optimized by leave-one-out cross-validation method using predicted residual error sum of square. The proposed model exhibited a high capability in simultaneous prediction of MOR and IBU concentrations in real samples. The results showed linear ranges of 1.33–33.29μg/mL (R2=0.9904) and 0.28–6.9μg/mL (R2=0.9902) for MOR and IBU respectively with low detection limits of 0.15 and 0.03μg/mL(S/N=5).

Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LC–MS/MS and its application to a bioequivalence study

Pritesh Contractor, Abhishek Gandhi, Gajendra Solanki, Priyanka A. Shah, Pranav S. Shrivastav

2017, 7(6): 417-422.

Abstract(45) PDF(1)

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An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D2) in human plasma using LC–MS/MS.After liquid-liquid extraction with n-hexane,ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C18 (100 mm×4.6 mm, 2.5μm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0 min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring(MRM).Entire data processing was done using Watson LIMS? software which provided excellent data integrity and high throughput with improved operational efficiency.The major advantage of this method includes higher sensitivity(0.10 ng/mL), superior extraction efficiency(≥83%)and small sample volume(100μL)for processing.The method was linear in the concentration range of 0.10–100 ng/mL for ergocalciferol.The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25 mg ergocalciferol capsules in 12 healthy subjects.

2017 Vol. 7, No. 6